Work

D‐amino acid oxidase as a chemogenetic tool for spatiotemporally controlled hydrogen peroxide production: The oxygen connection

Year: 2025

Type: article

Abstract: BACKGROUND AND PURPOSE: production may influence cellular oxygen levels. We set out to explore if DAO activation results in hypoxia, which in turn alters the activity of cellular oxygen sensors such as prolyl-4-hydroxylase domain enzymes, and thus the activation of the hypoxia-inducible factor (HIF) pathway. EXPERIMENTAL APPROACH: We analysed oxygen the consumption rate (OCR), pericellular oxygen concentration and HIF-1α stabilisation in the cytoplasm or mitochondrial matrix of HEK293 cells stably overexpressing DAO. The obtained experimental data were used to test whether a simple mathematical model can be used to predict oxygen levels. KEY RESULTS: For HEK293 cells expressing DAO in either the cytoplasm or mitochondrial matrix, we found a dose-dependent increase in OCR upon stimulating the DAO reaction, which resulted in a decreased pericellular oxygen concentration. Correspondingly, HIF-1α protein levels were stabilised over time, reflecting the biological cellular response to DAO-induced hypoxia. Furthermore, we provide a simplified diffusion model that allows users to input experimental parameters and assess the risk of hypoxic conditions in cell cultures. CONCLUSIONS AND IMPLICATIONS: High DAO activity can induce hypoxia because of increased oxygen consumption. Caution should be taken to prevent hypoxic conditions in cell culture experiments when using DAO. A provided spreadsheet is intended to help estimate oxygen levels in combination with OCR measurements.

Source: British Journal of Pharmacology

DOI: 10.1111/bph.70046